Previous work on the role of adenosine receptor subtypes as pro-angiogenic factors has largely been in vitro and has implicated the A2 receptors on vascular cells including endothelial and smooth muscle cells, as the mediators for the effect. Our in vivo data in the chicken chorioallantoic membrane (CAM) assay unexpectedly indicate that the A1 receptor is among the subtypes that stimulate angiogenesis. Furthermore, we have determined that the A1 adenosine receptor likely works through inflammatory, not vascular, cells by stimulating the release of vascular endothelial growth factor (VEGF) from monocytes/macrophages. We hypothesize that adenosine promotes angiogenesis at a number of different levels. A2 receptor stimulation triggers endothelial cell proliferation and release of VEGF from smooth muscle cells, while A1 receptor stimulation promotes angiogenesis through its effects on inflammatory cells, namely monocytes/macrophages. The A3 receptor may work through effects on mast cells. We have formulated three Specific Aims to test these hypotheses: Aim I): We will use novel subtype-selective ligands and allosteric enhancers of adenosine receptors to pharmacologically characterize the chicken, rat, mouse, and human adenosine receptors. The results of Aim I will guide the selection of ligand concentrations for subsequent aims. Aim II): We will use three models of angiogenesis, the CAM, rat thoracic aortic ring, and rat mesenteric models to demonstrate in vivo modulation of angiogenesis by adenosine agonists, antagonists, and allosteric enhancers. A fourth model, the ischemic rat hindlimb model, is in development and will be used to test the ability of pro-angiogenic ligands and enhancers to improve blood flow to an ischemic muscle bed. Aim III): We will use subtype-selective ligands on cultured cells to identify mechanisms of adenosine-stimulated angiogenesis. We will identify which adenosine receptor subtypes trigger endothelial cell proliferation and which stimulate cytokine release from smooth muscle cells, mast cells, and monocytes/macrophages. Our ultimate goal is therapeutic manipulation of angiogenesis.